Project Summary/Abstract Role of transcription in genomic stability Transcription is a two-edged sword with respect to its role in genomic stability. RNA polymerase (RNAP) can sensitively detect damage in DNA as it performs one-dimensional scanning, thus initiating the dedicated pathway of transcription-coupled repair (TCR) that removes lesions from transcribed strands of expressed genes. However, transcription can also be mutagenic. Some hot spots for mutations that have been implicated in carcinogenesis are localized in DNA sequences that can form non-canonical DNA structures. We wish to gain a mechanistic perspective on the contribution to genomic instability when translocating RNAP encounters such structures. We hypothesize that TCR can be mutagenic when it occurs near non-canonical DNA structures, bound complex ligands, or interstrand crosslinks. To test this hypothesis we will explore the signals that can elicit the TCR response, to determine whether TCR might be error-prone under some circumstances. Utilizing a well-established transcription assay with purified T7 RNAP or mammalian RNAPII on defined DNA substrates we will: (1) Characterize translocating RNAP at site-specific lesions and non-canonical DNA structures, including Z-DNA, H-DNA triplexes, triplet-repeat sequences, cruciform structures generated from palindromic sequences, and psoralen-mediated interstrand crosslinks. Effects of negative supercoiling as well as single and multiple rounds of transcription will be evaluated. The arrested RNAP, transcription bubble, and RNA/DNA hybrid will be mapped. (2) Determine compounding effects upon transcription of abasic sites or 8oxoGuanine, when introduced into noncanonical DNA structures. (3) Analyze effects on transcription of complex ligands specific for certain DNA sequences, including DNA binding proteins, such as ADAR1 on Z-DNA and topoisomerases trapped at lesion sites. Psoralen-conjugated triplex-forming oligonucleotides (TFOs) and peptide nucleic acid (PNA) will be utilized to explore novel aspects of these unique ligands for targeted gene alterations, as well as to learn how they affect RNAP translocation. Mechanistic information about TCR will be obtained by adding purified DNA repair proteins and/or transcription elongation factors (eg. CSB, TFIIH, XPG, MSH2-MSH3, MSH2-MSH6, TFIIS, and RecQ) to the transcription assays. The results from this project will enhance our understanding of the role of transcription and TCR in processing lesions and other abnormalities in DNA that have been implicated in carcinogenesis. Since transcription arrest generates a strong signal for apoptosis, the research may lead to novel modes of cancer chemotherapy, involving selective inhibition of TCR in target cells combined with administration of transcription-blocking drugs.